Project Summary The Src Homology 2 (SH2) domain is a unique domain that allows a protein to recognize and bind specific protein partners containing phosphotyrosine (pTyr) residues. One SH2-containing protein in particular, the Signal Transducer and Activator of Transcription 3 (STAT3), is overexpressed in a variety of hematological and solid malignancies and requires the use of its SH2 domain for activation and transcriptional activity. Targeted therapies aimed at the STAT3 SH2 domain could be a viable strategy to inhibit STAT3-dependant cancers, however work over the last two decades has largely resulted in molecules suffering from a lack of specificity, reduced cell permeability from the negatively charged phosphate group, and intracellular cleavage by cellular phosphatases. Because the pTyr residue confers several of these barriers, SH2 inhibitors could benefit greatly from a pTyr mimic. Our lab has previously studied a 3-residue pTyr-mimicking motif that demonstrated the ability to bind the Grb2 SH2 domain. Importantly, work in our lab has demonstrated that conferring conformational constraint to peptides incorporating this motif improved affinity and selectivity towards the target protein, as well as serum stability and cell permeability. Based on this work, our hypothesis is that this pTyr-mimicking motif can be used as a scaffold and incorporated into additional peptides for the potent and selective inhibition of different SH2 domain-containing proteins, including STAT3, providing a better pharmacologic profile to more selective peptide inhibitors. To accomplish this goal, we plan to utilize two parallel approaches. The first is a rational design approach in which we utilize known STAT3 SH2 binding sequences incorporating the motif in place of pTyr, and using thiol-mediated bisalkylation with different chemical linkers to create small libraries of uniquely constrained cyclic peptides. A simultaneous, more high throughput approach, will utilize a one-bead-one- compound library, varying the motif itself with unnatural amino acids to screen thousands of peptide combinations that provide the optimal torsional constraints required for achieving pTyr mimicry, as assessed through ability to capture an anti-pTyr antibody. This motif will then be applied to larger libraries (106 members) targeting the STAT3 SH2 domain directly. Peptides will be tested for STAT3 binding affinity, cell permeability, selectivity over other SH2 domains including the pro-apoptotic STAT1, STAT3 phosphorylation and transcriptional activity, and ultimately induction of STAT3-dependent cancer cell death. It is the goal of this proposal to develop a pTyr-mimicking, cell permeable peptide capable of binding the STAT3 SH2 domain for the induction of STAT3-dependent cancer cell death. Additionally, it is our hope that future work may utilize this strategy and optimized pTyr-mimicking motif in the development of selective inhibitors of other SH2 domains for the treatment of human disease.